Glucosyl stevia composition

ABSTRACT

Glucosyl  stevia  compositions are prepared from steviol glycosides of  Stevia rebaudiana  Bertoni. The glucosylation was performed by cyclodextrin glucanotransferase using the starch as source of glucose residues. The glucosyl  stevia  compositions were purified to &gt;95% content of total steviol glycosides. The compositions can be used as sweetness enhancers, flavors, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals.

RELATED APPLICATIONS

This application is a continuation-in-part application of and claims thebenefit of priority to:

U.S. patent application Ser. No. 14/040,986, filed on Sep. 30, 2013,which is a continuation-in-part application of U.S. patent applicationSer. No. 13/567,707 filed on Aug. 6, 2012, issued as U.S. Pat. No.8,647,844 on Feb. 11, 2014, which is a divisional application of U.S.patent application Ser. No. 13/029,263 filed on Feb. 17, 2011, issued asU.S. Patent No. 8,257,948 on Sep. 4, 2012;

U.S. patent application Ser. No. 14/040,986 is also acontinuation-in-part application of U.S. patent application Ser. No.13/656,868 filed on Oct. 22, 2012, issued as U.S. Pat. No. 8,669,077 onMar. 11, 2014, which is a divisional application of U.S. patentapplication Ser. No. 13/074,179 filed on Mar. 29, 2011, now U.S. Pat.No. 8,318,459 issued on Nov. 27, 2012, which is a continuation-in-partapplication of U.S. patent application Ser. No. 13/029,263 filed on Feb.17, 2011, issued as U.S. Pat. No. 8,257,948 on Sep. 4, 2012; and

U.S. patent application Ser. No. 14/519,403, filed Oct. 21, 2014, whichis a continuation of U.S. patent application Ser. No. 13/984,884, filedon Aug. 12, 2013, now U.S. Pat. No. 8,911,971, issued Dec. 16, 2014,which is a 371 of international application number PCT/US2011/035173,filed on May 4, 2011. The entire contents of each of the aforementionedpriority applications and patents is incorporated by reference herein.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to a process for producing a highly purified foodingredient from the extract of the Stevia rebaudiana Bertoni plant andits use in various food products and beverages.

Description of the Related Art

Nowadays sugar alternatives are receiving increasing attention due toawareness of many diseases in conjunction with consumption of high-sugarfoods and beverages. However many artificial sweeteners such as dulcin,sodium cyclamate and saccharin were banned or restricted in somecountries due to concerns on their safety. Therefore non-caloricsweeteners of natural origin are becoming increasingly popular. Thesweet herb Stevia rebaudiana Bertoni, produces a number of diterpeneglycosides which feature high intensity sweetness and sensory propertiessuperior to those of many other high potency sweeteners.

The above-mentioned sweet glycosides, have a common aglycon, steviol,and differ by the number and type of carbohydrate residues at the C13and C19 positions. The leaves of Stevia are able to accumulate up to10-20% (on dry weight basis) steviol glycosides. The major glycosidesfound in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%),and Rebaudioside C (1-2%). Other glycosides such as Rebaudioside B, D,E, and F, Steviolbioside and Rubusoside are found at much lower levels(approx. 0-0.2%).

Two major glycosides—Stevioside and Rebaudioside A, were extensivelystudied and characterized in terms of their suitability as commercialhigh intensity sweeteners. Stability studies in carbonated beveragesconfirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983)Stability studies of stevioside and Rebaudioside A in carbonatedbeverages. J. Agric. Food Chem. 31: 409-412.)

Steviol glycosides differ from each other not only by molecularstructure, but also by their taste properties. Usually stevioside isfound to be 110-270 times sweeter than sucrose, Rebaudioside A between150 and 320 times, and Rebaudioside C between 40-60 times sweeter thansucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside Ahas the least astringent, the least bitter, and the least persistentaftertaste thus possessing the most favorable sensory attributes inmajor steviol glycosides (Tanaka O. (1987) Improvement of taste ofnatural sweeteners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989)Stevia: steps in developing a new sweetener. In: Grenby T. H. ed.Developments in sweeteners, vol. 3. Elsevier Applied Science, London.1-43.)

Methods for the extraction and purification of sweet glycosides from theStevia rebaudiana plant using water or organic solvents are describedin, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938;5,972,120; 5,962,678; 7,838,044 and 7,862,845.

However, even in a highly purified state, steviol glycosides stillpossess undesirable taste attributes such as bitterness, sweetaftertaste, licorice flavor, etc. One of the main obstacles for thesuccessful commercialization of stevia sweeteners are these undesirabletaste attributes. It was shown that these flavor notes become moreprominent as the concentration of steviol glycosides increases (PrakashI., DuBois G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008)Development of rebiana, a natural, non-caloric sweetener. Food Chem.Toxicol., 46, S75-S82.)

On the other hand, replacing large amounts of sugar in the formulationsbrings up such problems as reduced mouthfeel, incomplete flavor profileetc. Therefore the application of high intensity low calorie sweetenershas to provide solutions to address these problems.

Thus, if a single composition will be able to deliver not onlysweetness, but also possess flavor enhancing properties and correct theincomplete mouthfeel associated with the elimination of sucrose fromfood and beverage formulations, it will certainly be advantageouscompared to other high intensity sweeteners known in the art.

Some of these undesirable properties can be reduced or eliminated bysubjecting steviol glycosides to the reaction of intermoleculartransglycosylation, when new carbohydrate residues are attached toinitial molecule at C13 and C19 positions. Depending on the number ofcarbohydrate residues in these positions the quality and potency of thecompounds taste will vary.

Pullulanase, isomaltase (Lobov S. V., Jasai R., Ohtani K., Tanaka O.Yamasaki K. (1991) Enzymatic production of sweet stevioside derivatives:transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965),β-galactosidase (Kitahata S., Ishikawa S., Miyata T., Tanaka O. (1989)Production of rubusoside derivatives by transglycosylation of variousβ-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextransaccharase (Yamamoto K., Yoshikawa K., Okada S. (1994) Effectiveproduction of glucosyl-stevioside by α-1,6-transglucosylation of dextrandextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used astransglycosylating enzymes, together with pullulan, maltose, lactose,and partially hydrolyzed starch, respectively, as donors of glycosidicresidues.

The transglucosylation of steviol glycosides was also performed byaction of cyclodextrin glucanotransferases (CGTase) produced by Bacillusstearothermophilus (U.S. Pat. Nos. 4,219,571, and 7,807,206) as a resultα-1,4-glucosyl derivatives were formed with degree of polymerization upto 10.

It was shown that the taste profile and sweetness power of glucosylderivatives are largely dependent on number of additional glucosylderivatives, i.e. the degree of polymerization of the α-1,4-glucosylchain. The increase in number of α-1,4-glucosyl residues improved thetaste quality but at the same time reduced the sweetness level (Tanaka,1987). The treatment of transglucosylated stevioside with β-amylaseresulted in a product consisting of mono- or di-α-1,4-glucosylderivatives (Tanaka, 1987).

However in such processes, the resulting product contains a high levelof initial unreacted (unmodified) glycosides (generally >20%) whichmakes it not compliant with regulatory requirements of less than 15%unreacted glycosides (α-Glucosyltransferase Treated Stevia, Japan'sSpecifications and Standards for Food Additives, VIII edition, 2009, p.257). Therefore additional steps for chromatographic separation ofunreacted steviol glycosides are used to reduce initial unreacted(unmodified) glycosides' content. However chromatographic separationtechniques generally involve high cost and are not suitable for largescale production.

It is noted also that many glucosyl stevia products contain up to 20%residual dextrins which do not possess significant functional propertiesand reduce the content of steviol glycosides in the product.

Therefore it is necessary to develop high purity products with anoptimal α-1,4-glucosyl chain length and low unreacted glycosides levelwhich will deliver the best combination of sweetness potency and flavorprofile.

SUMMARY OF THE INVENTION

The present invention is aimed to overcome the disadvantages of existingStevia sweeteners. The invention describes a process for producing ahigh purity food ingredient from the extract of the Stevia rebaudianaBertoni plant and use thereof in various food products and beverages asa sweetness and flavor modifier.

The invention, in part, pertains to an ingredient comprisingglucosylated derivatives of steviol glycosides of Stevia rebaudianaBertoni plant. The steviol glycosides are selected from the groupconsisting of stevioside, Rebaudioside A, Rebaudioside B, RebaudiosideC, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside X,dulcoside A, steviolbioside, rubusoside, as well as other steviolglycosides found in Stevia rebaudiana Bertoni plant and mixturesthereof.

The invention, in part, pertains to a process for producing aningredient containing glucosylated forms of stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, Rebaudioside X, dulcoside A, steviolbioside, rubusoside,as well as other steviol glycosides found in Stevia rebaudiana Bertoniplant. The process can be an enzymatic transglucosylation process usingCGTases produced by cultures of Bacillus stearothermophilus. The processmay include the step of additional enzymatic treatment by β-amylase orother enzymes. The process can also have the steps of decolorizing,desalting and removing maltooligosaccharides. The decolorizing can beperformed using activated carbon. The desalting can be performed bypassing through ion exchange resins and/or membrane filters. Removingthe maltooligosaccharides can be performed by passing throughmacroporous polymeric resin.

In the invention, Stevia extract commercialized by PureCircle (JiangXi)Co., Ltd. (China), containing stevioside (28-30%), Rebaudioside A(50-55%), Rebaudioside C (9-12%), Rebaudioside F (1-3%) and otherglycosides amounting to total steviol glycosides' content of at least95%, was used as a starting material. Alternatively stevia extracts withdifferent ratio of steviol glycosides as well as highly purified steviolglycosides such as Rebaudioside A, stevioside, Rebaudioside D,Rebaudioside X, rubusoside etc, may be used as starting materials.

The starting material was subjected to the enzymatic transglucosylationby action of cyclodextrin glycosyltransferase (CGTase) in the presenceof starch as a glucose donor. As a result α-1,4-glucosyl derivativeswere formed, in some embodiments with degree of polymerization up to 20.The formed derivatives were optionally subjected to treatment withβ-amylase or other enzymes to produce α-1,4-glucosyl derivativespossessing a specific degree of polymerization.

The oligosaccharides from obtained reaction mixture were removed byAmberlite XAD7 HP resin, and then decolorized, deionized, concentratedand spray dried.

The obtained products were applied in various foods and beverages assweeteners, sweetener enhancers, flavors and flavor modifiers, includingsoft drinks, ice cream, cookies, bread, fruit juices, milk products,baked goods and confectionary products.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows a high-performance liquid chromatographic chromatogram ofpurified transglucosylated Stevia extract, without β-amylase treatmentcontaining long-chain α-1,4-glucosyl-derivatives with up to nineα-1,4-glucosyl residues;

FIG. 2 shows a high-performance liquid chromatographic chromatogram ofpurified transglucosylated Stevia extract after β-amylase treatment withshort-chain (containing four or less α-1,4-glucosyl residues)derivatives of stevioside and Rebaudioside A.

FIG. 3 shows a high-performance liquid chromatographic (HPLC)chromatogram of β-amylase treated product containing mono- anddi-α-1,4-glucosyl-derivatives of steviol glycosides, as well as highlevel of unreacted steviol glycoside;

FIG. 4 shows a high-performance liquid chromatographic (HPLC)chromatogram of β-amylase treated product containing mono- anddi-α-1,4-glucosyl-derivatives of steviol glycosides, as well as lowlevel of unreacted steviol glycosides.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Stevia extract commercialized by PureCircle (JiangXi) Co., Ltd. (China),containing stevioside (28-30%), Rebaudioside A (50-55%), Rebaudioside C(9-12%), Rebaudioside F (1-3%) and other glycosides (hereinaftercollectively, “steviol glycosides”) amounting to total steviolglycosides content of at least 95%, was used as a starting material.Alternatively stevia extracts with different ratio of steviol glycosidesas well as highly purified steviol glycosides such as Rebaudioside A,stevioside, Rebaudioside D, Rebaudioside X, rubusoside etc, may be usedas starting materials.

In certain embodiments, the steviol glycosides may be replaced,partially or completely, by compounds from the group consisting of LuoHan Guo extract, Siraitia grosvenorii extract, mogrosides, mogrosideIIE, mogroside III, mogroside IV, mogroside V, mogroside VI,11-oxo-mogroside V, siamenoside I, grosmomoside I, as well as othermogrol or oxo-mogrol glycosides found in Siraitia grosvenorii plant andmixtures thereof.

The HPLC analysis of the raw materials and products was performed onAgilent

Technologies 1200 Series (USA) liquid chromatograph, equipped withZorbax-NH₂ (4.6×250 mm) column. The mobile phase was acetonitrile-watergradient from 80:20, v/v (0-2 min) to 50:50, v/v (2-70 min). A diodearray detector set at 210 nm was used as the detector.

The transglucosylation was accomplished by cyclomaltodextringlucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillusstearothermophilus St-88 (PureCircle Sdn Bhd Collection of IndustrialMicroorganisms—Malaysia). However, any other CGTase or enzyme possessingintermolecular transglucosylation activity may be applied as well. Theenzyme can be in a form of cell-free culture broth, concentrated liquidcell-free culture broth, spray dried or freeze dried cell-free culturebroth, or high purity protein. Free and immobilized enzyme preparationscan be used.

The activity of CGTase preparations was determined according to theprocedure described in Hale W. S., Rawlins L. C. (1951) Amylase ofBacillus macerans. Cereal Chem. 28, 49-58.

Starches of different origin may be used as donors of glucosyl unitssuch as, derived from wheat, corn, potato, tapioca, and sago.

Starch was subjected to partial hydrolysis (liquefaction) prior to thetransglucosylation reaction. The dextrose equivalent of the partiallyhydrolyzed starch can be in the range of about 10-25, preferably about12-16. Any enzyme capable of starch hydrolysis may be used forliquefaction, such as α-amylases, β-amylases etc. In one embodiment,CGTase and α-amylase mixtures as liquefying enzymes are preferred.

α-Amylase activity is expressed in Kilo Novo α-amylase Units (KNU). OneKNU is the amount of α-amylase which, under standard conditions (pH 7.1;37° C.), dextrinizes 5.26 g starch dry substance per hour.

The liquefaction mixture contains about 0.001-0.2 KNU, preferably about0.05-0.1 KNU of α-amylase per one unit of CGTase.

The use of α-amylase in liquefaction allows achieving higher throughputsin further activated carbon filtration. When the CGTase is used as theonly liquefying enzyme the filtration rate is approximately 10-15 L/hrper 1 m² of filter surface. In case of liquefaction enzyme mixture(comprising α-amylase and CGTase) the filtration rate is twice asfast—approximately 20-30 L/hr per 1 m² of filter surface.

The ratio of starch and CGTase in the liquefaction mixture is about0.1-0.5 units per one gram of starch, preferably about 0.2-0.4 units pergram.

The concentration of starch in liquefaction mixture is about 15-40%(wt/wt), preferably about 20-30%.

The liquefaction is conducted at about 70-90° C., or 75-80° C., duringabout 0.5-5 hours, for example, about 0.5 to 2 hours, and preferablyabout 1-2 hours.

After liquefaction, the reaction mixture is subjected to thermalinactivation of α-amylase at low pH conditions. The preferred pH rangefor inactivation is about pH 2.5 to pH 3.0 and preferred temperature isabout 95-105° C. The duration of thermal inactivation is about 5-10minutes.

After the inactivation, the pH of the reaction mixture is adjusted toabout pH 5.5-6.5 and the steviol glycosides are added to the mixture anddissolved. The preferred ratio of steviol glycosides to starch (kg ofsteviol glycosides per 1 kg of starch) is about 0.5-1.5, preferablyabout 0.8-1.2.

A second portion of CGTase preparation is added and thetransglucosylation reaction is conducted at temperature of between about5-125° C., such as 65° C., for about 1 to 168 hours, such as 24-48hours. The amount of the second portion of CGTase is about 0.2-4 unitsof CGTase per gram of solids, preferably about 0.5-1.2 units per gram ofsolids.

After the addition of the second portion of the CGTase preparation,additional steps may include optionally inactivating the enzyme(s) inthe reaction mixture; optionally decolorizing the reaction mixture; andoptionally concentrating and drying the reaction mixture to obtainglucosyl stevia composition. In certain embodiments, the glucosyl steviacomposition at this stage comprises steviol glycoside derivatives havingtwenty or less α-1,4-glucosyl residues.

Upon completion of transglucosylation reaction, further enzymatictreatment or treatments, and additional steps, can be used to arrive atthe desired degree of polymerization and unreacted glycosides in thecomposition.

Further enzymatic treatment can include the addition of amylase,β-amylase, maltase, glucoamylase, fructofuranosidase, glucosidase,glucanase, β-glucanase, transglucosidase, glucosyltransferase,fructosyltransferase, galactosyltransferase, lactase, galactosidase,cellulase, pullulanase, xylanase, mannanase, Maltogenase®, Fungamyl®,Novamyl®, Optimait®, or mixtures thereof, along with the substrate orsubstrates for the respective enzyme or enzymes utilized. The reactionmixture can be incubated for a period of time ranging from 0.0001 to 168hours, at a temperature ranging from 5-125° C.

Additional steps may include inactivating the enzymes in the reactionmixture by heat treatment; optionally decolorizing the reaction mixture;optionally removing non-diterpene compounds by contacting thedecolorized reaction mixture with macroporous adsorbent resin andsubsequently eluting adsorbed diterpene glycosides with alcohol oraqueous alcohol to result in a glycoside-containing eluate; optionallydesalting the glycoside-containing eluate with ion-exchange resins;optionally removing alcohol from the eluate, resulting in an aqueouseluate; optionally concentrating and drying the aqueous eluate to obtainthe dried glucosyl stevia composition, and optionally suspending thedried glucosyl stevia composition in aqueous alcohol, separating thecrystals from suspension and drying them to obtain the desired glucosylstevia composition.

The order of any of these steps may be changed depending on a variety offactors.

In certain embodiments, upon completion of transglucosylation reaction,about 30-50 units per gram of solids of β-amylase was added and thereaction was continued for about 12-16 hours at about 35-55° C.,preferably about 45° C. Soybean β-amylase was used in this stage forSamples 1a and 2a, while the β-amylase made in accordance with EXAMPLE 2was used for Samples 1b and 2b. However β-amylases derived from anyother source including barley, bacterial, fungal β-amylases and othersmay be used as well.

β-Amylase activity unit (1 AUN) is defined as the activity whichliberates 100 μg of reducing sugar (expressed by dextrose equivalent)per minute under the following conditions: 1 mL of enzyme solution ismixed with 5 mL of 1.2% starch solution (pH 5.5, M/20 Acetate Buffer)and kept for 20 min at 40° C.

The reaction was stopped by heating at about 95° C. for about 15 minutesto inactivate the enzymes, and the solution was treated with activatedcarbon, to obtain decolorized reaction mixture. The amount of activatedcarbon was about 0.02-0.4 grams per gram of solids, preferably about0.05-0.2 grams per gram of solids. Other appropriate decolorizingmethods, such as using ion exchange resins, membrane filtration usingultrafiltration, nanofiltration or reverse osmosis membranes, or othermethods known in the art can be used.

Non-diterpene compounds may optionally be removed using, for example, aplurality of sequentially connected columns packed with a macroporousadsorbent resin, followed by washing the columns with water, thenwashing with about 10-50% (v/v) ethanol, disconnecting the columns, andthen eluting each column individually with 30-100% ethanol.

The decolorized reaction mixture was desalted by passing through ionexchange resins, such as Amberlite FPC23 (H⁺ type) and Amberlite FPA51(OH⁻ type). Other appropriate decolorizing and desalting methods such asmembrane filtration or other methods known in the art can be used.

The desalted reaction mixture was further concentrated by vacuumevaporator and dried by means of a spray dryer. Other appropriateconcentrating and drying methods, such as membrane filtration, freezedrying, or other methods known to art can be used.

The dried powder was suspended in aqueous alcohol. The powder to aqueousalcohol ratio (wt/vol) was 1:1 to 1: 20, preferably 1:3 to 1:10. Theaqueous alcohol contained 0-50% (vol), preferably 1-10% water. Thesuspension is agitated at 30-100° C., preferably 50-85° C. during 1-24hours, preferably 2-15 hours. Then the suspended solids are separated bymeans of filtration. Any other technique known in the art suitable forseparating suspended solids from liquid such as centrifugation,decanting, etc. can be used. The obtained solids are dried in rotarydrum vacuum drier. Any other dryer known in the art may be used as well.Alternatively the separated solids may be dissolved in water, evaporatedfrom traces of alcohol and spray dried.

The alcohols employed in the invention may be selected from the groupconsisting of alkanols, and are preferably selected from the groupincluding methanol, ethanol, n-propanol, 2-propanol, 1-butanol, and2-butanol, or mixtures thereof.

In certain embodiments, the resulting product contains low levelnon-modified glycosides, short-chain (containing four or less, or two orless, α-1,4-glucosyl residues) derivatives and a mixture ofmaltooligosaccharides (Samples 1a and 1b). As used herein, theexpressions “low level non-modified glycosides” or “low level unreactedglycosides” shall refer to glycoside levels of less than about 20%, andpreferably less than about 15%, on an anhydrous basis. In someembodiments, an unreacted glycoside level of about 12%, about 10% oreven lower can be attained using this method.

In order to prepare a product with higher content of total sweetglycosides (the sum of glucosylated and non-glucosylated glycosides),the maltooligosaccharides were removed using Amberlite XAD7 HP prior tothe desalting treatment. The steviol glycosides and their glucosylatedderivatives were adsorbed on the resin and subsequently eluted byaqueous ethanol. The resulted aqueous ethanol eluate, containingglucosyl steviol glycosides, was subsequently decolorized and desaltedas described above and the glycosides solution, after the evaporation ofeluting solvent, was powdered by spray drying. The dried powder wassuspended in aqueous alcohol and processed as described above to removeunmodified (unreacted) steviol glycosides (Sample 2b). The resultingproduct contains low level non-modified glycosides, and short-chain(containing four or less, or two or less α-1,4-glucosyl residues)derivatives (Samples 2a and 2b). The embodiments of the inventionexemplified by Samples 1a, 1b, 2a and 2b are free or substantially freeof higher glucosylated derivatives having more than 4 or more than 2glucosyl residues. In accordance with this invention, the highlypurified glucosyl stevia composition preferably comprises greater thanabout 25% by weight di-, tri- and tetraglucosyl Rebaudioside A, andgreater than about 9% by weight tri- and tetraglucosyl steviosides. Inanother embodiment, the highly purified glucosyl stevia compositioncomprises greater than about 50% by weight mono-, and diglucosyl steviolglycosides.

Using a similar process as for Sample 2a, with exclusion of theβ-amylase treatment stage, a product containing non-modified glycosidesand long chain α-1,4-glucosyl-derivatives (with up to nineα-1,4-glucosyl residues) was prepared (Sample 3).

As a control, a commercial β-amylase treated product containingnon-modified glycosides, and short-chain (containing two or lessα-1,4-glucosyl residues) derivatives was used (Sample 4).

The composition of the samples is summarized in Tables 1a and 1b, inwhich Samples 1a and 2a made using the processes described above containfour or less α-1,4-glucosyl residues, and Samples 1b and 2b made usingthe processes described above contain two or less α-1,4-glucosylresidues.

TABLE 1a Composition of glucosyl steviol glycosides samples containing 4or fewer α-1,4-glucosyl residues Content, % Sample Sample Sample SampleCompounds 1a 2a 3 4 Stevioside 2.5 3.0 3.1 9.5 Rebaudioside C 0.9 1.01.0 0.4 Rebaudioside A 5.2 6.1 6.0 2.8 Monoglucosyl-stevioside (StevG1)11.0 13.2 7.4 34.9 Monoglucosyl-Rebaudioside A 14.6 17.5 11.1 6.3(RebAG1) Diglucosyl-stevioside (StevG2) 10.4 12.4 8.4 26.4Diglucosyl-Rebaudioside A 15.6 18.6 9.6 — (RebAG2)Triglucosyl-stevioside (StevG3) 5.8 7.0 6.3 — Triglucosyl-Rebaudioside A7.9 9.5 7.7 — (RebAG3) Tetraglucosyl-stevioside (StevG4) 3.7 4.4 5.6 —Tetraglucosyl-Rebaudioside A 2.9 3.4 6.1 — (RebAG4) Higher glucosylatedderivatives 22.7 — Total content of glycosides 80.5 96.1 95.0 80.3

The sensory assessment of samples was carried using aqueous solutions,with 20 panelists. Based on overall acceptance the most desirable andmost undesirable samples were chosen. The results are shown in Table 2a.

TABLE 2a Sensory assessment of samples in water system Judgment Sample1a Sample 2a Sample 3 Sample 4 Most 5 11 1 2 desirable Most 1 0 7 12undesirable Sweetness 150 160 120 150 power Comments Sweet, light,Sweet, light, Sweet, Sweet, soft, round, soft, round, slightly slightlypleasant, pleasant, bitter, bitter, almost similar similar toastringent, astringent, to sucrose, no no lingering slight sucrose, nolingering aftertaste, lingering lingering aftertaste, sweetnessaftertaste, aftertaste, sweetness onset sweetness sweetness onset is ismoderate onset is slow onset rapid is rapid

As apparent from the results in Table 2a, the sweetness quality of theSamples 1a and 2a was rated as most superior. Overall the samples withshort-chain (containing four or less α-1,4-glucosyl residues)derivatives (Sample 1a, and Samples 2a) possessed better taste profilescompared to samples with long-chain glucosyl derivatives (Sample 3) andtwo or less α-1,4-glucosyl residues short-chain derivatives (Sample 4).

Samples 1a and 2a show comparable sweetness power (150-160 times sweetercompared to a 5% sucrose solution) with control Sample 4 (150 times);however their flavor profile was clearly superior to the control sample.

A similar analysis was done for Samples 1b and 2b, which contain two orfewer α-1,4-glucosyl residues. Sample 5 was prepared in accordance withEXAMPLE 12.

TABLE 1b Composition of glucosyl steviol glycosides samples containing 2or fewer α-1,4-glucosyl residues Content, % Sample Sample Sample SampleCompounds 1b 2b 3 5 Stevioside 2.4 3.2 3.1 13.2 Rebaudioside C 0.7 1.01.0 3.0 Rebaudioside A 5.6 7.5 6.1 12.3 Monoglucosyl-stevioside (StevG1)16.2 21.9 7.5 22.2 Monoglucosyl-Rebaudioside A 20.9 28.1 11.2 22.4(RebAG1) Diglucosyl-stevioside (StevG2) 10.1 13.6 8.5 8.9Diglucosyl-Rebaudioside A 13.8 18.6 9.7 11.4 (RebAG2) Higherglucosylated derivatives 1.3 1.7 48.8 1.8 Total content of unreacted 8.711.7 10.2 28.5 glycosides Total content of glycosides 71.0 95.5 95.895.3

The sensory assessment of samples was carried using aqueous solutions,with 20 panelists. Based on overall acceptance the most desirable andmost undesirable samples were chosen. The results are shown in Table 2b.

TABLE 2b Sensory assessment of samples in water system Judgment Sample1b Sample 2b Sample 3 Sample 5 Most 6 10 1 3 desirable Most 1 0 12 7undesirable Sweetness 150 160 110 160 power Comments Sweet, light,Sweet, light, Sweet, Sweet, soft, round, soft, round, slightly slightlypleasant, pleasant, bitter, bitter, almost similar to astringent,astringent, similar to sucrose, no slight lingering slight sucrose, nolingering aftertaste, lingering lingering aftertaste, sweetnessaftertaste, aftertaste, sweetness onset sweetness sweetness onset is ismoderate onset is slow onset rapid is rapid

As apparent from the results in Table 2b, the sweetness quality of theSamples 1b and 2b was rated as most superior. Overall the samples withshort-chain (containing two or less α-1,4-glucosyl residues) derivativesand low level of unreacted glycosides (Samples 1b and 2b) possessedbetter taste profiles compared to samples with long-chain glucosylderivatives (Sample 3) and short-chain (containing two or lessα-1,4-glucosyl residues) derivatives and high level of unreactedglycosides (Sample 5).

Samples 1b and 2b show comparable sweetness power (150-160 times sweetercompared to a 5% sucrose solution) with control Sample 5 (160 times);however their flavor profile was clearly superior to the control Sample5.

The compositions can be used as sweetness enhancers, flavors, flavorenhancers and sweeteners in various food and beverage products.Non-limiting examples of food and beverage products include carbonatedsoft drinks, ready to drink beverages, energy drinks, isotonic drinks,low-calorie drinks, zero-calorie drinks, sports drinks, teas, fruit andvegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcoholbeverages, powdered beverages, bakery products, cookies, biscuits,baking mixes, cereals, confectioneries, candies, toffees, chewing gum,dairy products, flavored milk, yoghurts, flavored yoghurts, culturedmilk, soy sauce and other soy base products, salad dressings,mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products,bottled and canned foods, tabletop sweeteners, fruits and vegetables.

Additionally the compositions can be used in drug or pharmaceuticalpreparations and cosmetics, including but not limited to toothpaste,mouthwash, cough syrup, chewable tablets, lozenges, vitaminpreparations, and the like.

The compositions can be used “as-is” or in combination with othersweeteners, flavors and food ingredients.

Non-limiting examples of sweeteners include steviol glycosides,stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideD, Rebaudioside E, Rebaudioside F, Rebaudioside X, dulcoside A,steviolbioside, rubusoside, as well as other steviol glycosides found inStevia rebaudiana Bertoni plant and mixtures thereof, stevia extract,Luo Han Guo extract, mogrosides, high-fructose corn syrup, corn syrup,invert sugar, fructooligosaccharides, inulin, inulooligosaccharides,coupling sugar, maltooligosaccharides, maltodextins, corn syrup solids,glucose, fructose, maltose, sucrose, lactose, aspartame, saccharin,sucralose, sugar alcohols, and combinations thereof.

Non-limiting examples of flavors include lemon, orange, fruity, banana,grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cottoncandy, vanilla flavors.

Non-limiting examples of other food ingredients include flavors,acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilisers, thickeners, gelling agents, and combinationsthereof.

The following examples illustrate various embodiments of the invention.It will be understood that the invention is not limited to thematerials, proportions, conditions and procedures set forth in theexamples, which are only illustrative.

EXAMPLE 1 Preparation of CGTase

A strain of Bacillus stearothermophilus St-88 was inoculated in 2,000liters of sterilized culture medium containing 1.0% starch, 0.25% cornextract, 0.5% (NH₄)9SO₄, and 0.2% CaCO₃ (pH 7.0-7.5) at 56° C. for 24hrs with continuous aeration (2,000 L/min) and agitation (150 rpm). Theobtained culture broth was filtered using Kerasep 0.1 μm ceramicmembrane (Novasep, France) to separate the cells. The cell-free permeatewas further concentrated 2-fold on Persep 10 kDa ultrafilters (Orelis,France). The activity of the enzyme was determined according to Hale,Rawlins (1951). A crude enzyme preparation with activity of about 2unit/mL was obtained.

EXAMPLE 2

Preparation of β-amylase

A strain of Bacillus polymyxa St-3504 was inoculated in 2,000 liters ofsterilized culture medium containing 1.0% starch, 0.5% peptone, 0.5%corn extract, 0.5% NaCl, 0.02% MnSO₄ and 0.1% CaCO₃ (pH 7.0-7.5) at 32°C. for 24 hrs with continuous aeration (2,000 L/min) and agitation(150rpm). The obtained culture broth was filtered using Kerasep 0.1 μmceramic membrane (Novasep, France) to separate the cells. 10% of glucosewas added to the cell-free permeate which was further concentrated onPersep 10 kDa ultrafilters (Orelis, France) and dried using Alpha 1-4LSC freeze drier unit (Christ, Germany) to obtain a powder with 20,000AUN/g activity. 62 -Amylase activity unit (1 AUN) was defined as theactivity which liberates 100 μg of reducing sugar (expressed by dextroseequivalent) per minute under the following conditions: 1 mL of enzymesolution is mixed with 5 mL of 1.2% starch solution (pH 5.5, M/20Acetate Buffer) and kept for 20 min at 40° C.

EXAMPLE 3 Preparation of Short-Chain Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units soybean β-amylase (#1500S, NagaseChemtex Corp., Japan) was added to reaction mixture. The reaction wascontinued for another 12 hours. The obtained reaction mixture was heatedat 95° C. for 15 minutes to inactivate the enzymes. 20 grams ofactivated carbon was added and the mixture was heated to 75° C. and heldfor 30 minutes. The mixture was filtered and the filtrate was dilutedwith water to 5% solids content and passed through columns packed withAmberlite FPC23 (H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. Thedesalted solution was concentrated at 60° C. under vacuum, and driedinto a powder form using laboratory spray dryer. 196 grams of productwas obtained (Sample 1a).

EXAMPLE 4 Preparation of Highly Purified Short-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units soybean β-amylase (#1500S, NagaseChemtex Corp., Japan) was added to reaction mixture. The reaction wascontinued for another 12 hours. The obtained reaction mixture was heatedat 95° C. for 15 minutes to inactivate the enzymes. 20 grams ofactivated carbon was added and the mixture was heated to 75° C. and heldfor 30 minutes. The mixture was filtered and the filtrate was dilutedwith water to 5% solids content and passed through columns each packedwith 4000 mL Amberlite XAD 7HP macroporous adsorbent resin. The columnswere washed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol.The adsorbed glycosides were eluted with 50% ethanol. Obtained eluatewas passed through columns packed with Amberlite FPC23 (H⁺) andAmberlite FPA51 (OH⁻) ion exchange resins. The ethanol was evaporatedand the desalted and decolorized water solution was concentrated at 60°C. under vacuum, then dried into a powder form using laboratory spraydryer. 151 grams of product was obtained (Sample 2a).

EXAMPLE 5 Preparation of Highly Purified Long-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20grams of activated carbon was added and the mixture was heated to 75° C.and held during 30 min. The mixture was filtered and the filtrate wasdiluted with water to 5% solids content and passed through columns eachpacked with 4000 mL Amberlite XAD 7HP macroporous adsorbent resin. Thecolumns were washed with 5 volumes of water and 2 volumes of 20% (v/v)ethanol. The adsorbed glycosides were eluted with 50% ethanol. Obtainedeluate was passed through columns packed with Amberlite FPC23 (H⁺) andAmberlite FPA51 (OH⁻) ion exchange resins. The ethanol was evaporatedand the desalted and decolorized water solution was concentrated at 60°C. under vacuum, then dried into a powder form using laboratory spraydryer. Approximately 165 grams of product was obtained (Sample 3).

EXAMPLE 6 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03%) were blended anddissolved completely in water (up to 100%) and pasteurized. Glucosylstevia compositions were represented by Samples 1a, 2a, and 3, obtainedaccording to EXAMPLES 3, 4, and 5, respectively; and Sample 4 was acommercial β-amylase treated product (containing only mono- anddi-α-1,4-glucosyl-derivatives of steviol glycosides).

The sensory evaluations of the samples are summarized in Table 3. Thedata show that the best results can be obtained by using the high purityshort-chain glucosyl stevia compositions (containing four or lessα-1,4-glucosyl residues) derivatives (Samples 1a and 2a). Particularlythe drinks prepared with Samples 1a and 2a exhibited a rounded andcomplete flavor profile and mouthfeel.

TABLE 3 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel No. 1a High quality sweetness, Clean, almost noFull pleasant taste similar to bitterness, no aftertaste sucrose,rounded and balanced flavor No. 2a High quality sweetness, Clean, nobitterness and Full pleasant taste similar to no aftertaste sucrose,rounded and balanced flavor No. 3 High quality sweetness, Clean, almostno Almost pleasant taste almost bitterness, no aftertaste acceptablesimilar to sucrose, rounded and balanced flavor No. 4 Sweet, licoricenotes Slight bitterness and Not aftertaste acceptable

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

EXAMPLE 7 Low-Calorie Carbonated Beverage

A carbonated beverage according to formula presented below was prepared.

Ingredients Quantity, % Sucrose 5.5 Cola flavor 0.340 ortho-Phosphoricacid 0.100 Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018Rebaudioside A 0.003 Glucosyl stevia composition 0.05 Carbonated waterto 100

The sensory properties were evaluated by 20 panelists. The results aresummarized in Table 4.

TABLE 4 Evaluation of low-calorie carbonated beverage samples Number ofpanelists detected the attribute Sample Sample Sample Sample No. 1a No.2a No. 3 No. 4 Taste attribute Bitter taste 0 0 2 20 Astringent 1 0 3 20taste Aftertaste 1 0 2 20 Comments Quality of Clean Clean Clean Bitteraftertaste sweet taste (19 of 20) (20 of 20) (17 of 20) (5 of 20)Overall Satisfactory Satisfactory Satisfactory Satisfactory evaluation(18 of 20) (20 of 20) (15 of 20) (3 of 20)

The above results show that the beverages prepared using Samples 1a and2a possessed the best organoleptic characteristics.

EXAMPLE 8 Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%)and different glucosyl stevia compositions (0.03%) were kneaded well indough-mixing machine. The obtained dough was molded and baked in oven at200° C. for 15 minutes. Glucosyl stevia compositions were by representedby Samples 1a, 2a, and 3, obtained according to EXAMPLES 3, 4, and 5,respectively; with Sample 4 being a commercial β-amylase treated product(containing only mono- and di-α-1,4-glucosyl-derivatives of steviolglycosides).

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing four or less α-1,4-glucosyl residues)derivatives (Samples 1a and 2a). The panelists noted rounded andcomplete flavor profile and mouthfeel in cookies prepared with Samples1a and 2a.

EXAMPLE 9 Yoghurt

Different glucosyl stevia compositions (0.03%) and sucrose (4%) weredissolved in low fat milk. Glucosyl stevia compositions were byrepresented by Samples 1a, 2a, and 3, obtained according to EXAMPLES 3,4, and 5, respectively; with Sample 4 being a commercial β-amylasetreated product (containing only mono- and di-α-1,4-glucosyl-derivativesof steviol glycosides). After pasteurizing at 82° C. for 20 minutes, themilk was cooled to 37° C. A starter culture (3%) was added and themixture was incubated at 37° C. for 6 hours then at 5° C. for 12 hours.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing four or less α-1,4-glucosyl residues)derivatives (Samples 1a and 2a). The panelists noted rounded andcomplete flavor profile and mouthfeel in samples prepared with Samples1a and 2a.

EXAMPLE 10 Preparation of Short-Chain Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according toEXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns packed with Amberlite FPC23(H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. The desaltedsolution was concentrated at 60° C. under vacuum, and dried into apowder form using laboratory spray dryer. The dried powder was suspendedin 5 volumes of 95% aqueous ethanol. The suspension was agitated at 80°C., during 12 hours. Then the suspended solids were separated byfiltration. The obtained solids were dried in vacuum dryer at 90° C.during 5 hours. 170 grams of product was obtained (Sample 1b).

EXAMPLE 11

Preparation of Highly Purified Short-Chain Glucosyl Stevia Composition100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according toEXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The columns were washedwith 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol.

Obtained eluate was passed through columns packed with Amberlite FPC23(H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. The ethanol wasevaporated and the desalted and decolorized water solution wasconcentrated at 60° C. under vacuum, then dried into a powder form usinglaboratory spray dryer. The dried powder was suspended in 5 volumes of95% aqueous ethanol. The suspension was agitated at 80° C., during 12hours. Then the suspended solids were separated by filtration. Theobtained solids were dried in vacuum dryer at 90° C. during 5 hours. 121grams of product was obtained (Sample 2b).

EXAMPLE 12 Preparation of Highly Purified Short-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according to

EXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The columns were washedwith 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol. Obtained eluate was passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The ethanol was evaporated and the desaltedand decolorized water solution was concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. 154 grams ofproduct was obtained (Sample 5).

EXAMPLE 13 Preparation of Long-Chain Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20grams of activated carbon was added and the mixture was heated to 75° C.and held during 30 min. The mixture was filtered and the filtrate wasconcentrated at 60° C. under vacuum, then dried into a powder form usinglaboratory spray dryer. 197 grams of product was obtained (Sample 6).

EXAMPLE 14 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03%) were blended anddissolved completely in water (up to 100%) and pasteurized. Glucosylstevia compositions were represented by Samples 1b, 2b, 3, 5 and 6,obtained according to EXAMPLES 10, 11, 5, 12, and 13 respectively.

The sensory evaluations of the samples are summarized in Table 5. Thedata show that the best results can be obtained by using the high purityshort-chain glucosyl stevia compositions (containing two or lessα-1,4-glucosyl residues and low unreacted steviol glycosides) (Samples1b and 2b). Particularly the drinks prepared with Samples 1b and 2bexhibited a rounded and complete flavor profile and mouthfeel.

TABLE 5 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel No. 1b High quality sweetness, Clean, almost noFull pleasant taste similar to bitterness, no aftertaste sucrose,rounded and balanced flavor No. 2b High quality sweetness, Clean, nobitterness and Full pleasant taste similar to no aftertaste sucrose,rounded and balanced flavor No. 3 High quality sweetness, Clean, almostno Almost pleasant taste almost bitterness, no aftertaste acceptablesimilar to sucrose, rounded and balanced flavor No. 5 Sweet, licoricenotes Slight bitterness and Not aftertaste acceptable

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

EXAMPLE 15 Low-Calorie Carbonated Beverage

A carbonated beverage according to formula presented below was prepared.

Ingredients Quantity, % Sucrose 5.5 Cola flavor 0.340 ortho-Phosphoricacid 0.100 Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018Rebaudioside A 0.003 Glucosyl stevia composition 0.05 Carbonated waterto 100

The sensory properties were evaluated by 20 panelists. The results aresummarized in Table 6.

TABLE 6 Evaluation of low-calorie carbonated beverage samples Number ofpanelists detected the attribute Sample Sample No. 1b No. 2b Sample No.3 Sample No. 5 Taste attribute Bitter taste 0 0 10 12 Astringent 1 0 1515 taste Aftertaste 1 0 13 18 Comments Quality of Clean Clean CleanBitter aftertaste sweet taste (18 of 20) (20 of 20) (14 of 20) (10 of20) Overall Satisfactory Satisfactory Satisfactory Satisfactoryevaluation (19 of 20) (20 of 20) (11 of 20) (9 of 20)

The above results show that the beverages prepared using Samples 1b and2b possessed the best organoleptic characteristics.

EXAMPLE 16 Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%)and different glucosyl stevia compositions (0.03%) were kneaded well indough-mixing machine. The obtained dough was molded and baked in oven at200° C. for 15 minutes. Glucosyl stevia compositions were represented bySamples 1b, 2b, 3, and 5, obtained according to EXAMPLES 10, 11, 5, and12, respectively.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing two or less α-1,4-glucosyl residues)derivatives (Samples 1b and 2b). The panelists noted rounded andcomplete flavor profile and mouthfeel in cookies prepared with Samples1b and 2b.

EXAMPLE 17 Yoghurt

Different glucosyl stevia compositions (0.03%) and sucrose (4%) weredissolved in low fat milk. Glucosyl stevia compositions were representedby Samples 1b, 2b, 3, and 5, obtained according to EXAMPLES 10, 11, 5,and 12, respectively. After pasteurizing at 82° C. for 20 minutes, themilk was cooled to 37° C. A starter culture (3%) was added and themixture was incubated at 37° C. for 6 hours then at 5° C. for 12 hours.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing two or less α-1,4-glucosyl residues)derivatives (Samples 1b and 2b). The panelists noted rounded andcomplete flavor profile and mouthfeel in samples prepared with Samples1b and 2b.

COMPARATIVE EXAMPLE 1 Preparation of Highly Purified Short-ChainGlucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to the procedure described above were added,and the liquefaction of starch was carried out at 80° C. for about onehour to dextrose equivalent about 15.

After cooling to 65° C., the pH was adjusted to pH 6.0 with sodiumhydroxide solution. 100 g stevia extract produced by PureCircle(JiangXi) Co., Ltd. (China), containing stevioside 29.2%, Rebaudioside A54.3%, Rebaudioside C 9.0%, Rebaudioside F (1.7%) and other glycosidesamounting to total steviol glycosides content of about 96.4% was addedto liquefied starch and stirred until a homogeneous solution wasobtained. 200 units of CGTase was added to the solution and the mixturewas held at a temperature of 65° C. for 24 hours under continuousagitation.

Then the temperature was reduced to 45° C., and 8,000 units soybeanβ-amylase (#1500S, Nagase Chemtex Corp., Japan) was added to reactionmixture. The reaction was continued for another 12 hours. The obtainedreaction mixture was heated at 95° C. for 15 minutes to inactivate theenzymes. 20 grams of activated carbon was added and the mixture washeated to 75° C. and held for 30 minutes. The mixture was filtered andthe filtrate was diluted with water to 5% solids and was passed throughcolumns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51 (OH⁻) ionexchange resins and then through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The macroporous resincolumns were washed with 5 volumes of water and 2 volumes of 20% (v/v)ethanol. The adsorbed glycosides were eluted with 50% ethanol. Theethanol of obtained eluate was evaporated and concentrated at 60° C.under vacuum, then dried into a powder form using laboratory spraydryer. 114 grams of product was obtained (Sample 7).

The Sample 7 composition was analyzed using HPLC, and its sensoryassessment along with other samples (Samples 1a, 2a, 3 and 4) wascarried out using aqueous solutions, with 20 trained panelists.

Composition of Glucosyl Steviol Glycoside Samples

Content, % Compounds Sample 1a Sample 2a Sample 3 Sample 4 Sample 7Stevioside 2.5 3.0 3.1 9.5 17.1 Rebaudioside C 0.9 1.0 1.0 0.4 4.2Rebaudioside A 5.2 6.1 6.0 2.8 27.7 Monoglucosyl-stevioside (StevG1)11.0 13.2 7.4 34.9 13.9 Monoglucosyl-Rebaudioside A (RebAG1) 14.6 17.511.1 6.3 17.1 Diglucosyl-stevioside (StevG2) 10.4 12.4 8.4 26.4 5.9Diglucosyl-Rebaudioside A (RebAG2) 15.6 18.6 9.6 — 7.2Triglucosyl-stevioside (StevG3) 5.8 7.0 6.3 — 1.1Triglucosyl-Rebaudioside A (RebAG3) 7.9 9.5 7.7 — 1.4Tetraglucosyl-stevioside (StevG4) 3.7 4.4 5.6 — —Tetraglucosyl-Rebaudioside A (RebAG4) 2.9 3.4 6.1 — — Higherglucosylated derivatives — — 22.7 — — Unreacted glycosides (Stev +RebC + RebA) 8.6 10.1 10.1 12.7 49.0 Total content of glycosides 80.596.1 95.0 80.3 95.6

Sensory Assessment of Samples in Water System

Judgment Sample 1a Sample 2a Sample 3 Sample 4 Sample 7 Most desirable 611 1 2 0 Most undesirable 0 0 4 6 10 Comments Sweet, light, Sweet,light, Sweet, Sweet, Sweet, soft, round, soft, round, slightly slightlybitter, pleasant, pleasant, bitter, bitter, astringent, almost similarsimilar to astringent, astringent, lingering to sucrose, no no lingeringslight aftertaste, sucrose, no lingering aftertaste, lingering sweetnesslingering aftertaste, sweetness aftertaste, onset is slow aftertaste,sweetness onset is sweetness sweetness onset is moderate onset is slowonset is rapid rapid

COMPARATIVE EXAMPLE 2 Preparation of Highly Purified Short-ChainGlucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to the procedure described above were added,and the liquefaction of starch was carried out at 80° C. for about onehour to dextrose equivalent about 15. After cooling to 65° C., the pHwas adjusted to pH 6.0 with sodium hydroxide solution. 100 g steviaextract produced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according tothe procedure described above was added to reaction mixture. Thereaction was continued for another 12 hours. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzymes.20 grams of activated carbon was added and the mixture was heated to 75°C. and held for 30 minutes. The mixture was filtered and the filtratewas diluted with water to 5% solids and was passed through columnspacked with Amberlite FPC23 (H⁺) and Amberlite FPA51 (OH⁻) ion exchangeresins and then through columns each packed with 4000 mL Amberlite XAD7HP macroporous adsorbent resin. The macroporous resin columns werewashed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol. Theadsorbed glycosides were eluted with 50% ethanol. The ethanol ofobtained eluate was evaporated and concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. The driedpowder was suspended in 5 volumes of 95% aqueous ethanol. The suspensionwas agitated at 80° C., during 12 hours. Then the suspended solids wereseparated by filtration. The obtained solids were dried in vacuum dryerat 90° C. during 5 hours. 67 grams of product was obtained (Sample 8).

The Sample 8 composition was analyzed using HPLC, and its sensoryassessment along with other samples (Samples 1b, 2b, 3, and 5 asdescribed above) was carried out using aqueous solutions, with 20trained panelists.

Composition of glucosyl Steviol Glycoside Samples

Content, % Compounds Sample 1b Sample 2b Sample 3 Sample 5 Sample 8Stevioside 2.4 3.2 3.1 13.2 20.79 Rebaudioside C 0.7 1.0 1.0 3.0 6.15Rebaudioside A 5.6 7.5 6.1 12.3 32.46 Monoglucosyl-stevioside (StevG1)16.2 21.9 7.5 22.2 12.95 Monoglucosyl-Rebaudioside A (RebAG1) 20.9 28.111.2 22.4 18.57 Diglucosyl-stevioside (StevG2) 10.1 13.6 8.5 8.9 1.69Diglucosyl-Rebaudioside A (RebAG2) 13.8 18.6 9.7 11.4 2.40 Higherglucosylated derivatives 1.3 1.7 48.8 1.8 — Total content of unreactedglycosides 8.7 11.7 10.2 28.5 59.4 Total content of glycosides 71.0 95.595.8 95.3 95.0

Sensory Assessment of Samples in Water System

Judgment Sample 1b Sample 2b Sample 3 Sample 5 Sample 8 Most desirable 710 1 2 0 Most undesirable 0 0 5 4 11 Comments Sweet, light, Sweet,light, Sweet, Sweet, Sweet, soft, round, soft, round, slightly slightlybitter, pleasant, pleasant, bitter, bitter, astringent, almost similarsimilar to astringent, astringent, lingering to sucrose, no slightslight aftertaste, sucrose, no lingering lingering lingering sweetnesslingering aftertaste, aftertaste, aftertaste, onset is slow aftertaste,sweetness sweetness sweetness sweetness onset is onset is onset is slowonset is rapid rapid moderate

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereof, and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

1. A process for producing a glucosyl mogrol glycoside composition,comprising the steps of: adding starch into water to form a starchsuspension; adding a mixture of α-amylase and cyclodextringlucaonotransferase (CGTase) into the starch suspension and incubatingfor about 0.5 to 2 hours at about 75-80° C., resulting in a liquefiedstarch suspension; inactivating the α-amylase by low pH heat treatment;adding mogrol glycosides into the liquefied starch suspension, resultingin a reaction mixture; adding a second batch of CGTase into the reactionmixture and incubating the reaction mixture for about 1 to 168 hours atabout 5-125° C.
 2. The process according to claim 1, further includingthe steps of: adding one or several additional enzymes selected from thegroup consisting of: maltase, fructofuranosidase, glucosidase,glucanase, β-glucanase, transglucosidase, glucosyltransferase,fructosyltransferase, galactosyltransferase, lactase, galactosidase,cellulase, pullulanase, xylanase, mannanase, or mixtures thereof, andincubating the additional enzymes in the reaction mixture for about0.0001-168 hours at about 5-125° C.; wherein the glucosyl mogrolcomposition comprises mogrol glycoside derivatives having twenty or lessα-1,4-glucosyl residues.
 3. (canceled)
 4. The process of claim 1,wherein the mixture of α-amylase and CGTase contains about 0.05-0.1 KNUof α-amylase per one unit of CGTase.
 5. The process according to claim1, wherein the weight of added mogrol steviol glycosides is about equalto that of the starch.
 6. The process according to claim 1, furthercomprising the step of adding to the liquefied starch suspensioncompounds selected from the group consisting of stevioside, rebaudiosideA, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E,rebaudioside F, rebaudioside X, dulcoside A, steviolbioside, rubusoside,as well as other steviol glycosides found in Stevia rebaudiana plant andmixtures thereof.
 7. The process according to claim 1, wherein the addedmogrol glycosides are selected from the group consisting of Luo Han Guoextract, Siraitia grosvenorii extract, mogrosides, mogroside IIE,mogroside III, mogroside IV, mogroside V, mogroside VI, 11-oxo-mogrosideV, siamenoside grosmomoside I, as well as other mogrol or oxo-mogrolglycosides found in Siraitia grosvenorii plant and mixtures thereof. 8.The process according to claim 1, wherein the CGTase is produced bycultures of Bacillus stearothermophilus.
 9. The process according toclaim 1, wherein the second batch of CGTase has about 0.2-4 units ofCGTase per gram of solids.
 10. The process according to claim 1, whereinthe second batch of CGTase has about 0.5-1.2 units of CGTase per gram ofsolids.
 11. The process according to claim 2, wherein the β-amylase isproduced from a source selected from the group consisting of soybeans,barley, fungi, and bacteria.
 12. The process according to claim 2,wherein the β-amylase is added at about 30-50 units per gram of totalsolids, and the treatment is carried out at a temperature of about40-60° C., for a duration of about 3-16 hours.
 13. The process accordingto claim 2, wherein after the additional enzyme treatment, theglucosylated derivatives of mogrol glycosides have four or lessα-glucosyl residues.
 14. The process according to claim 2, wherein afterthe additional enzyme treatment, the glucosylated derivatives of mogrolglycosides have two or less α-glucosyl residues.
 15. The processaccording to claim 2, wherein after the additional enzyme treatment, theglucosylated derivatives of mogrol glycosides have only one α-glucosylresidue.
 16. The process according to claim 2, further comprising thestep of adding a substrate of the enzyme to the reaction mixture. 17.The process of claim 2, further comprising inactivating the additionalenzymes in the reaction mixture by heat treatment after incubating thereaction mixture.
 18. The process of claim 2, further comprising a stepof decolorizing the reaction mixture.
 19. (canceled)
 20. The process ofclaim 2, further comprising a step of desalting the reaction mixturewith ion-exchange resins.
 21. (canceled)
 22. The process of claim 2,further comprising a step of concentrating and drying the reactionmixture to obtain a dried glucosyl mogrol glycoside composition.
 23. Theprocess of claim 22, further comprising a step of suspending the driedglucosyl mogrol composition in aqueous alcohol to obtain crystals,separating the crystals from suspension and drying them to obtain theglucosyl mogrol glycoside composition,
 24. The process of claim 18,wherein the decolorizing is performed using activated carbon.
 25. Theprocess according to claim 18, wherein the decolorizing is performedusing ion exchange resins or membranes, said membranes being selectedfrom the group consisting of ultrafiltration, nanofiltration, andreverse osmosis membranes.
 26. (canceled)
 27. The process according toclaim 20, wherein the desalting is performed by passing the reactionmixture through columns packed with ion exchange resins or membranes,said membranes being selected from the group consisting ofultrafiltration, nanofiltration, and reverse osmosis membranes. 28.(canceled)
 29. A composition comprising the glucosyl mogrol glycosidecomposition made by the process of claim 1, and an additional sweeteningagent selected from the group consisting of: stevia extract, steviolglycosides, stevioside, rebaudioside A, rebaudioside B, rebaudioside C,rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside X,dulcoside A, steviolbioside, rubusoside, other steviol glycosides foundin Stevia rebaudiana plant and mixtures thereof, Luo Han Guo extract,mogrosides, high-fructose corn syrup, corn syrup, invert sugar,fructooligosaccharides, inulin, inulooligosaccharides, coupling sugar,maltooligosaccharides, maltodextins, corn syrup solids, glucose,fructose, maltose, sucrose, lactose, aspartame, saccharin, sucralose,sugar alcohols, and a combination thereof
 30. A flavor compositioncomprising the glucosyl mogrol glycoside composition made by the processof claim 1, and an additional flavoring agent selected from the groupconsisting but not limited to: lemon, orange, fruity, banana, grape,pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cottoncandy, vanilla, and a combination thereof.
 31. A food ingredientcomprising the glucosyl mogrol glycoside composition made by the processof claim 1, and an additional food ingredient selected from the groupconsisting of: acidulants, organic and amino acids, coloring agents,bulking agents, modified starches, gums, texturizers, preservatives,antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, anda combination thereof.
 32. A food, beverage, cosmetic or pharmaceuticalproduct comprising the glucosyl mogrol glycoside composition made by theprocess of claim
 1. 33. The process of claim 1, further comprising astep of inactivating the enzyme in the reaction mixture. 34-35.(canceled)